Role of divalent cations in the resumption of meiosis of rat oocytes.

Resumption of meiotic maturation was induced in follicle-enclosed rat-oocytes by treatment with the divalent cationophore A23187 (10(-5)M). However, the same effect was attained by incubation in Ca++-deficient medium, even in the presence of EDTA or EGTA (1mM). The stability of the first polar body was increased under Ca++-deficient conditions. Neither the ionophore nor Ca++-deficient medium interfered with the spontaneous maturation of isolated oocytes of the rat. The experiments with cultured follicles suggest that redistribution of divalent cations may participate in the physiological control of meiosis in mammalian oocytes.     

Calcium effects on progesterone accumulation and oocyte maturation in cultured follicles of Rana pipiens

Pituitary homogenates (FPH) provoke a cascade of responses in the amphibian ovarian follicle, culminating in progesterone biosynthesis and oocyte maturation (GVBD). Calcium may play an important role as an intracellular second messenger in regulating these physiological responses. Experiments were carried out on cultured, isolated follicles of Rana pipiens to assess the effects of varying extracellular calcium on follicular progesterone accumulation and oocyte maturation. In hormonally unstimulated follicles, an increase in extracellular Ca2+ alone produced a significant increase in progesterone in methanol extracts of follicles after 4 hours of culture, and in some cases also provoked oocyte maturation assessed after 24 hours of culture. In no case did elevated Ca2+ alone stimulate maximal progesterone accumulation as compared with FPH-stimulated follicles, although the time-course of accumulation was similar. The calcium ionophore, A-23187, similarly increased progesterone accumulation in a dose-dependent manner when introduced in amphibian Ringer's (1.35 mM Ca2+), but inhibited progesterone elevation caused by increasing calcium concentrations in the culture media and FPH stimulation. Depleting free calcium from the culture medium with graded doses of the chelator EGTA decreased FPH-induced progesterone accumulation and inhibited FPH- and progesterone-induced GVBD. The calcium channel blocker, verapamil, also inhibited FPH-induced progesterone accumulation and GVDB in a dose-dependent manner, while having no effect on progesterone-induced meiotic resumption. These data strongly implicate intracellular calcium levels regulating progesterone production by ovarian follicle cells and subsequent oocyte maturation.     

Birth of common marmoset (Callithrix jacchus) offspring derived from in vitro-matured oocytes in chemically defined medium

Optimization of oocyte culture conditions is a crucial aspect of reproductive biology and technology. In the present study, maturation of germinal vesicle-stage marmoset oocytes were evaluated in the following media: Waymouth medium, Waymouth medium containing porcine follicular fluid (pFF) (Waymouth-pFF medium), and porcine oocyte medium (POM). Oocytes cultured in Waymouth-pFF medium had higher maturation rates to the metaphase II stage than those cultured in Waymouth medium (36.1% vs. 24.8%, respectively, P < 0.05), indicating the suitability of this medium for culturing marmoset oocytes. Hence, maturation of marmoset oocytes cultured in POM was subsequently evaluated. The rate of maturation to the metaphase I stage was significantly higher and degradation rates were significantly lower in oocytes cultured in POM than those cultured in Waymouth medium. In addition, three offspring were successfully obtained after transfer of embryos matured in chemically defined medium. Therefore, we concluded that POM was suitable for marmoset oocyte culture. Furthermore, this was apparently the first report of marmoset offspring derived from oocytes cultured in chemically defined medium.     

Ultrastructural localisation of calcium deposits in pig oocytes maturing in vitro: effects of verapamil

The culture of pig oocytes in the presence of the calcium channel blocker verapamil (0.02 mM) resulted in the blocking of meiosis at the metaphase I stage, and only a small fraction (about 28%) of the oocytes were able to continue their maturation to the stage of metaphase II. Hence, meiotic maturation in pig oocytes is a calcium-dependent process. After isolation of the pig oocytes from their follicles, the intracellular calcium deposits in the oocyte and granulosa cells, detectable using the combined oxalate-pyroantimonate method, are depleted. The amount of calcium deposits in the oocyte and granulosa cells increased during oocyte meiotic maturation in vitro, especially in the nucleus, mitochondria, vacuoles and cytoplasm. The replenishment of calcium deposits is significantly changed under the effect of verapamil. The increase in calcium deposits in the oocyte nucleus was delayed, a much larger amount of deposits was formed in the mitochondria, and the amount of deposits in the vacuoles was demonstrably smaller. A significant peak in the accumulation of calcium deposits was observed in the cytoplasm of verapamil-treated oocytes after 16 h of in vitro culture. We propose that an altered pattern in the replenishment of calcium deposits can disturb intracellular signalling and prevent the exit of oocytes from the metaphase I stage.     

Regulation of parthenogenetic activation of metaphase II mouse oocytes by pyruvate

We report that parthenogenetic activation (pronuclear formation) is induced during in vitro culture of recently ovulated (13-14 hr post-hCG) mouse oocytes in pyruvate deficient medium. Pronuclear formation occurred when oocytes were cultured in medium containing 1/10X (Pyr-) or lower concentrations of pyruvate but failed to occur either in oocytes cultured in the presence of 0.47 mM (1X, Pyr+) or 1/2X pyruvate or in oocytes cultured in the absence of pyruvate but with cumulus cells. Pronuclear formation was evident within 8 hr of culture and completed by 16 hr and remained intact during continuous culture in Pyr- medium. Transfer of pronuclear oocytes to Pyr+ medium resulted in pronuclear membrane disassembly and further parthenogenetic development. A similar incidence of parthenogenetic activation occurred when recently ovulated oocytes were cultured in the presence of cycloheximide but not following ethanol or hyaluronidase treatment. However, both ethanol and hyaluronidase induced pronuclear formation in in vivo aged oocytes. Results suggest that the type of activation induced varies with the age of the oocyte and the nature of the stimulus. Amino acid uptake ([35S]methionine) by oocytes was unaffected by Pyr- culture whereas incorporation into protein was markedly inhibited. Gel electrophoretic analysis of labeled egg extracts revealed a marked inhibition of egg protein synthesis after 4 hr of culture in Pyr-. The occurrence of a cortical reaction was monitored by binding of fluorescent labeled lectin to the oocyte surface. A cortical reaction occurred in response to ethanol treatment of freshly ovulated and in vivo aged oocytes cultured in Pyr+ medium but not in pronucleate oocytes induced by Pyr- culture. Suppression of ethanol-induced cortical reaction by Pyr- culture was restored following transfer of oocytes to Pyr+ medium. Results demonstrate that nuclear events as well as plasma membrane events can be simply regulated by controlling the amount of energy substrate available to the germ cell. Effects of Pyr- culture in inducing pronuclear formation appear to be mediated in a large part via inhibition of protein synthesis.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Effect of inhibition of synthesis of inducible nitric oxide synthase-derived nitric oxide by aminoguanidine on the in vitro maturation of oocyte-cumulus complexes of cattle

The aim of the present study was to investigate the effects of inhibition of the enzyme inducible nitric oxide synthase (iNOS) by aminoguanidine (AG) on the in vitro maturation of oocyte-cumulus cell complex(es) (COC) of cattle. COC were cultured with different concentrations of AG (0, 1, 10, and 100mM) for 24h. In Experiment 1, the extent of cumulus complex expansion, nuclear maturation status and plasma membrane integrity of oocytes and cumulus cells from each treatment were assessed. Nitrate/nitrite (NO(3)(-)/NO(2)(-)) concentrations were determined in culture medium by the Griess method. Addition of different concentrations of AG to maturation medium promoted a dose-response inhibitory effect on cumulus expansion (P<0.05). Addition of 1 and 10mM AG to IVM medium did not affect plasma membrane integrity of oocytes or nuclear maturation rates (P>0.05), but it did reduce plasma membrane integrity in cumulus cells. One hundred millimolar inhibited pre-metaphase I (pre-MI) to metaphase II (MII) transition, promoted plasma membrane damage in oocytes (P<0.05), and increased NO(3)(-)/NO(2)(-) concentration when compared to controls (P<0.05). To evaluate if this effect was reversible, 10(-5)M sodium nitroprusside (SNP, NO donor) was added, only in the treatment with 100mM AG that inhibited the nuclear maturation. However, association of 10(-5)M SNP to 100mM AG did not reverse the effects of AG, but increased NO(3)(-)/NO(2)(-)concentration (P<0.05). In Experiment 2, the effect of different AG concentrations on cytoplasmic maturation in vitro was assessed based on cortical granule migration, and embryonic development. There was a dose effect on cortical granule migration rate, in which 1mM AG (83.9+/-6.2%) did not differ from control oocytes (83.6+/-8.2%; P>0.05), but 10mM partially inhibited migration (3.8+/-6.4%) and 100mM totally inhibited migration (P<0.05). SNP (10(-5)M) did not revert this inhibitory effect on cortical granules migration in oocytes treated with 100mM AG. Only those concentrations that did not inhibit IVM were used to assess cleavage and blastocyst development. Addition of 10mM AG to IVM medium reduced (73.0+/-8.1%, 15.0+/-8.9%; P<0.05) cleavage and blastocyst development, respectively when compared with controls (89.1+/-3.4%, 37.6+/-7.3%, respectively), but did not differ, (P>0.05), from the group treated with 1mM AG (80.9+/-8.4%, 41.5+/-10.5%, respectively). The results from the present study demonstrate that NO derived from iNOS affects the in vitro maturation of bovine COC, modulating the viability of cumulus cells and of oocyte, the progression of meiosis after GVBD, the migration of cortical granules, and cleavage and blastocyst development.     

Comparison of three in vitro culture systems for maturation of early preantral mouse ovarian follicles

The aim of this study was to compare three different culture systems for in vitro follicular growth and oocyte maturation in ovarian follicles of mice in order to assess the technique with the optimal growth and improved rate of meiotic maturation. The three systems tested were culture under oil, on a hydrophobic membrane and on agar respectively. Early preantral follicles were cultured for 12 days in alpha-MEM GlutaMAX medium. Follicular growth, oocyte meiotic maturation, oocyte extrusion, atresia and estradiol production were analysed. Follicular development showed two phases in the three systems, with slow growth before day 5 and subsequent acceleration. The percentage of follicles transferred into oocyte maturation medium was significantly higher after culture under oil. The proportion of oocytes that achieved nuclear maturation (metaphase II) was higher when follicles were cultured under oil or on a hydrophobic membrane than on agar. Our results support the use of culture under oil for in vitro follicular growth from the early preantral stage in order to obtain metaphase II oocytes. Fertilization ability of these oocytes and the capacity to obtain healthy mice in a reproducible manner warrants further investigation.     

Carbohydrate and amino acid requirements and ammonia production of rabbit follicular oocytes matured in vitro.

Rabbit follicular oocytes were cultured at 37 °C for 18–24 h in a basic salt medium containing 0.4% bovine serum albumin (BSA), carbohydrates and amino acids in various combinations. Osmolarity of the medium was maintained at 308 mOsm. The carbohydrates, pyruvate, lactate and glucose were all about equally beneficial, but not essential for rabbit oocyte maturation. Glutamine and proline, but not methionine or phenylalanine stimulated oocyte development. Glutamine stimulated more follicular oocytes to develop to prophase and metaphase II than did any of the three carbohydrates tested alone or in combination. Ammonia production after 24 h of culture was highest in media containing glutamine (15.2 μg/ml) but this was not inhibitory to maturation. Negligible amounts of ammonia were found with the other amino acids added. With 0, 0.08, 0.4, 2, 10 and 50 mM of glutamine in the basic salt medium, plus 0.4 % BSA, but without carbohydrates, 30, 73, 70, 71, 59 and 45 % of the follicular oocytes developed to the prophase or metaphase II stage. It is concluded that the optimum level of glutamine ranged from 0.08 to 2 mM and that no carbohydrate need be added to the medium for culturing oocytes when glutamine is included.     

Energy substrates and the completion of spontaneous meiotic maturation

This study was carried out to examine how different combinations of pyruvate and glucose affect spontaneous meiotic maturation of cumulus-cell-enclosed mouse oocytes (CEO) to metaphase II (MII). Most experiments used an open system in which oocytes were cultured in 1 ml medium in plastic tubes. Initial experiments examined the dose response effects of pyruvate or glucose alone in the presence or absence of 2 mM glutamine. When medium lacked both pyruvate and glucose, more than 91% of the oocytes died in glutamine-free medium during 15 h of culture; viability was restored with the addition of glutamine, but only 11% of the CEO reached MII. In the absence of glutamine, 62-68% of oocytes completed maturation in 0.23-2.3 mM pyruvate, while 44-60% MII was observed in 0.55-27.8 mM glucose. The addition of glutamine to these cultures had a general suppressive effect on the completion of maturation. When glucose was added to pyruvate-containing cultures, the combination of 1 mM pyruvate/5.5 mM glucose was most effective in supporting maturation (about 90% MII), with little effect of glutamine. No further increase in maturation was observed when glucose was increased five-fold (to 27.8 mM). The positive effect of glucose was in part attributed to stimulation of glycolysis and increased production of pyruvate, since a reduced culture volume (8 microl), which allows the accumulation of secreted pyruvate, improved maturation in glucose-containing, but not pyruvate-containing, medium, and FSH, which stimulates glycolysis, increased progression to MII in glucose-containing, but not pyruvate-containing, medium. Yet these results also suggest that glucose has a beneficial effect on maturation apart from simple provision of pyruvate. The pyruvate effect was directly on the oocyte, because denuded oocytes responded more effectively than CEO to this energy substrate. The highest percentage of MII oocytes (96-97%) occurred in microdrop cultures containing glucose but lacking glutamine. These results indicate that glutamine supports oocyte viability but is not an adequate energy source for the completion of spontaneous meiotic maturation and may be detrimental. In addition, while pyruvate and glucose alone can each support meiotic progression of CEO to MII, optimal maturation requires the provision of both substrates to the culture medium when a large volume (1 ml) is used. It is concluded that careful attention to specific energy substrate supplementation and culture volume is important to optimise spontaneous meiotic maturation in vitro.     

Requirement for, and patterns of, pyruvate and glutamine metabolism in the domestic dog oocyte in vitro

Supplementation of energy substrates to culture medium is essential for resumption and completion of meiosis in vitro for many mammalian species. Objectives were to study the dog oocyte, specifically the influences of pyruvate and glutamine on maturation and the utilization of these two substrates at various developmental stages and incubation times. Ovarian oocytes (n=681) were obtained from spayed bitches and cultured for 48 hr in TCM 199 medium containing various concentrations of pyruvate (0-2.5 mM) and glutamine (0-4 mM) before being assessed for nuclear status. For analyzing metabolic activity, 259 dog oocytes were cultured for 0, 12, 24, 36, or 48 hr, assessed for pyruvate and glutamine metabolism using the hanging drop method and then evaluated for nuclear status. Neither pyruvate nor glutamine had influence (P > 0.05) on oocyte maturation in vitro (IVM). However, both culture interval and meiotic status influenced pyruvate uptake (P < 0.05). Specifically, pyruvate uptake declined as the oocyte progressed from the germinal vesicle (GV) to metaphase II (MII) stage. Glutamine oxidation decreased as culture duration progressed (P < 0.05). In summary, pyruvate or glutamine is not required to promote successful IVM of dog oocytes. But, both substrates are being metabolized, and in patterns different to the domestic cat, another carnivore species. Pyruvate played an important role earlier in the maturational process, and less glutamine was oxidized as the oocyte neared nuclear maturation. These variations emphasize the importance of defining species specificities in carnivores before expecting consistently successful IVM/IVF.     

Effects of prolactin on intracellular stored calcium in the course of bovine oocyte maturation in vitro

At present there are divergent opinions as to the role of prolactin (PRL) in the mechanisms of meiotic regulation in mammals. We investigated the effects of bovine PRL (bPRL) on bovine oocyte maturation in different culture systems and varying levels of intracellular stored calcium ([Ca2+]is) in the oocytes. Cumulus-oocyte complexes (COC) were incubated in TCM 199 containing either 10% fetal calf serum (FCS) in the absence (System 1) or presence (System 2) of FSH and estradiol, or 6 mg/mL bovine serum albumin (BSA) in the presence of FSH and estradiol (System 3). Levels of [Ca2+]is in oocytes were determined by using the fluorophore chlortetracycline. The addition of 50 ng/mL bPRL to different culture media increased the percentage of oocytes at telophase I and metaphase II stages (Systems 1 and 2) and/or decreased the percentage of oocytes with degenerated chromosomes (Systems 1 and 3). Compared with the control, lower levels of [Ca2+]is were observed in oocytes cultured for 2.5 h in those systems in which bPRL decreased the rate of oocytes with degenerated chromosomes (1.27+/-0.11 vs. 1.67+/-0.09 arbitrary units (AU) in System 1, P<0.001 and 1.27+/-0.12 vs. 1.52+/-0.04 AU in System 3, P<0.001). These findings show that the effects of bPRL on bovine oocyte maturation depend on the composition of the culture system and that the decline in the rate of oocytes with degenerated chromosomes in response to bPRL may be the result of the decrease in [Ca2+ ]is levels at early stages of oocyte maturation.     

Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development

The objective was to evaluate the effects of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin on the nuclear and cytoplasmic maturation of bovine oocytes in the presence of follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22h in the presence of follicular cells (control with cells) and Ang II, IGF-I or insulin (treatments), or in the absence of follicular cells (control without cells). Using these five groups, Experiment 1 was conducted with and without the addition of gonadotrophins. Only oocytes in the Ang II group resumed meiosis at rates (88.2+/-1.8% and 90.7+/-4.3% for oocytes cultured in the absence or presence of LH/FSH, respectively) similar to those observed for oocytes cultured in the absence of follicular cells (89.7+/-0.3% and 92.6+/-2.6%; P<0.01). In Experiment 2, the effect of Ang II alone and in combination with IGF-I or insulin on oocyte maturation for 7h (germinal vesicle breakdown), 12h (metaphase I) and 22h (metaphase II) was evaluated in a design similar to that of the first experiment. Ang II plus IGF-I or insulin induced the resumption of meiosis, irrespective of the presence of gonadotrophins (P<0.01). Experiment 3 used groups similar Experiment 2 to determine the rate of subsequent embryo development, using fetal calf serum (FCS) in the culture medium. The COCs were cultured in maturation medium for 1h (1+23h), 12h (12+12h) or 24h in the presence of follicular cells and the respective treatments and for the remaining period in the absence of follicular cells to complete 24h. In Experiment 4, BSA was used in lieu of serum in the maturation medium in a 12+12h maturation system. Oocytes matured using the 12+12h system with BSA or FCS in the presence of Ang II+IGF-I had higher rates of blastocyst formation than the other treatments (P<0.05). In conclusion, Ang II reversed the inhibitory effect of follicular cells on nuclear maturation of bovine oocytes, irrespective of the presence of gonadotrophins, IGF-I and insulin. However, oocyte cytoplasmatic maturation (i.e., subsequent embryo development), was higher when Ang II and IGF-I were present in the maturation medium containing follicular cells cultured for 12+12h.     

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.     

Effects of beta-endorphin and Naloxone on in vitro maturation of bovine oocytes

Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.     

Initiation of meiotic maturation in Xenopus laevis oocytes by the combination of divalent cations and ionophore A23187.

Meiotic maturation was induced in Xenopus laevis oocytes when the external Ca++ or Mg++ ion concentration was raised above 5 mM in the presence of the ionophore. Ionophore-divalent cation-induced maturation appears to be due to the stimulation of the oocyte itself. Cytoplasm of responding oocytes induced maturation when microinjected into ovarian oocytes. Cycloheximid, an inhibitor of progesterone-induced maturation, inhibited the maturational response induced by the ionophore and divalent cations. Ethidium bromide, an inhibitor of the follicular response to human chorionic gonadotropin, had no effect. The possible roles that Ca++ and Mg++ may play in the initiation of maturation are discussed.     

Activation of pig oocytes by intracytoplasmic injection of strontium and barium

Ovulated mouse oocytes are activated by exposure to culture medium containing Sr2+ or Ba2+ or by intracytoplasmic injection of the divalent cations. It is known that in vitro matured pig oocytes are activated by the intracytoplasmic injection of Ca2+. In this study, we examined the effect of exposure and of intracytoplasmic injection of Sr2+ or Ba2+ on in vitro matured pig oocytes (MII-oocytes). When MII-oocytes were exposed to the medium containing divalent cations, no oocytes were activated. However, in the case of oocytes that were injected with Sr2+, Ba2+ and Ca2+, at 6 h after injection, 64%, 71% and 86% of the oocytes had been released from MII-arrest, and 51%, 67% and 84% formed female pronuclei, respectively. The initial transient in intracellular Ca2+ concentration ([Ca2+]i) was measured by the Ca2+ indicator dye fluo-4 dextran. Microinjection of Sr2+, Ba2+ or Ca2+ induced a rapid elevation of [Ca2+]i. The exocytosis of cortical granules was examined by staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin. After an injection of divalent cations, a release of cortical granules was observed in the oocytes. Maturation promoting factor (MPF) activity declined to a low level after 6 h in all the oocytes injected with divalent cations. To test their developmental ability, injected oocytes were treated with cytochalasin B and then cultured for 168 h in NCSU23 medium. After 168 h, 29% (Sr2+), 29% (Ba2+) and 51% (Ca2+) of the oocytes had developed to the blastocyst stage. These results indicate that intracytoplasmic injection of Sr2+ and Ba2+, like that of Ca2+, induces in vitro matured pig oocytes to be released from MII-arrest and leads them into a series of events related to oocyte activation.     

The ability to develop an activity that transfers histones onto sperm chromatin is acquired with meiotic competence during oocyte growth.

Following fertilization, the oocyte remodels the sperm chromatin into the male pronucleus. As a component of this process, during meiotic maturation, oocytes develop an activity that transfers histones onto sperm DNA. To further characterize this activity, we tested whether oocytes at different stages of growth could, upon entry into metaphase of maturation, transfer histones onto sperm DNA, as judged by chromatin morphology and immunocytochemistry. Meiotically competent growing oocytes, which spontaneously enter metaphase upon culture, transferred histones onto sperm chromatin, whereas incompetent oocytes did not, even when treated with okadaic acid to induce germinal vesicle breakdown (GVBD) and chromosome condensation. When incompetent oocytes were cultured until they acquired the ability to undergo GVBD, only a small proportion also developed histone-transfer activity during maturation. However, this proportion significantly increased when the oocytes were cultured as granulosa-oocyte complexes. The failure of histone-transfer activity to develop in incompetent oocytes treated with okadaic acid was not linked to low H1 kinase activity nor rescued by injected histones. Because competent, but not incompetent, oocytes produce natural calcium oscillations, incompetent oocytes were exposed to SrCl2. One-third of treated oocytes produced at least one Ca2+ oscillation and, following insemination, the same proportion transferred histones onto sperm DNA. Histone transfer did not occur in oocytes pretreated with the Ca2+ chelator, BAPTA-AM. These results indicate that the ability to develop histone-transfer activity is acquired by growing oocytes near the time of meiotic competence, that it is separable from this event, and that it may be regulated through a Ca2+-dependent process.     

Role of cyclic adenosine monophosphate (cAMP) in vitro on bovine oocyte maturation

This experiment attempted to determine the effect of cAMP on maturation of bovine oocytes in chemically-defined, serum-free medium. Cumulus-oocyte complexes were incubated in modified DME/Ham F-12 medium containing dbcAMP at 0 (control), 10(-6), 10(-4) and 10(-2) M. After 18 and 24 hours of culture, the percentage of oocyte maturation between 0 (control) and 10(-2) M dbcAMP-treated groups were significant. Some oocytes were cultured with dbcAMP (10(-2) M) for 6, 12 and 24 hours followed by incubation in control medium to test the reversibility of inhibition or of any harmful effect of dbcAMP. The inhibitory effect of 10(-2) M dbcAMP on bovine oocyte maturation was reversed by transferring cumulus-oocyte complexes to the control medium. In addition, forskolin (0.12 and 0.24 mM) was effective (P < 0.01) in preventing the resumption of meiosis. The cAMP content of oocytes cultured with forskolin was not increased, although cumulus cells responded to forskolin with an increase in cAMP content. These results indicate that elevated levels of cAMP in the culture medium are important in regulating resumption of meiosis of bovine oocytes in vitro.     

In the absence of protein, estradiol suppresses meiosis of porcine oocytes in vitro

The effect of estradiol on the spontaneous maturation of porcine oocytes was investigated. Cumulus-enclosed (intact) and cumulus-free (denuded) oocytes were cultured in the presence of estradiol-17 beta (0 to 10 microgram/ml) in a chemically defined bicarbonate-buffered medium that contained either dextran or BSA, or in a complex Hepes-buffered medium that was supplemented with serum. After 24 hr, chromatin spreads were prepared and meiotic maturation was scored. The biochemical integrities of the cumulus cells were assessed by determination of the estradiol and progesterone content of spent media after culture of intact oocytes in the presence of 0.5 X 10(-6) M testosterone and 10 microgram/ml follicle-stimulating hormone. Estradiol did not significantly affect the onset of maturation of either intact or denuded oocytes that were cultured in medium containing either BSA or serum. In serum-supplemented medium, however, the progression of maturation beyond metaphase I was significantly affected by the steroid in a dose-dependent manner. The steroid significantly inhibited the release from meiotic arrest of both types of oocyte cultured in medium supplemented with dextran. Supplementation of all media with testosterone and FSH significantly stimulated the synthesis of estradiol by the cumulus cells, compared with that of control groups. The synthesis of progesterone, however, was significantly stimulated by testosterone and FSH only in the BSA and serum-supplemented media. It is concluded that exogenous estradiol has the capacity to arrest meiosis in vitro but that this capacity can only be expressed if no exogenous protein(s) is present. In the absence of exogenous protein, progesterone synthesis by the adherent cumulus cells is minimal.